The primary event of sexual development in mammals is the development of the gonadal sex from a bipotential and undifferentiated gonad into either testes or ovaries. This process, known as sex determination, is triggered by the SRY gene (Sex-determining Region, Y chromosome). Evidence that Sry was sex determining initially came from the microinjection of a 14.6 kb genomic DNA sequence containing the mouse Sry gene into chromosomally female embryos. The resulting transgenic mice developed phenotypically as males [Koopman, P., J. Gubbay, N. Vivian, P. Goodfellow, and R. Lovell-Badge, Male development of chromosomally female mice transgenic for Sry. Nature, 1991. 351(6322): p. 117-21]. Sry belongs to the Sox (Sry-box) family, whose members are characterised by a common HMG (high mobility group) DNA-binding motif [Laudet, V., D. Stehelin, and H. Clevers, Ancestry and diversity of the HMG box superfamily. Nucleic Acids Res, 1993. 21(10): p. 2493-501; Wegner, M., From head to toes: the multiple facets of Sox proteins. Nucleic Acids Res, 1999. 27(6): p. 1409-20]. Sox genes have been documented in a wide range of developmental processes, including neurogenesis (Sox2, 3 and 10) [Hargrave, M., E. Wright, J. Kun, J. Emery, L. Cooper, and P. Koopman, Expression of the Sox11 gene in mouse embryos suggests roles in neuronal maturation and epithelio-mesenchymal induction. Dev Dyn, 1997. 210(2): p. 79-86; Rex, M., D. A. Uwanogho, A. Orme, P. J. Scotting, and P. T. Sharpe, cSox21 exhibits a complex and dynamic pattern of transcription during embryonic development of the chick central nervous system. Mech Dev, 1997. 66(1-2): p. 39-53; Uwanogho, D., M. Rex, E. J. Cartwright, G. Pearl, C. Healy, P. J. Scotting, and P. T. Sharpe, Embryonic expression of the chicken Sox2, Sox3 and Sox11 genes suggests an interactive role in neuronal development. Mech Dev, 1995. 49(1-2): p. 23-36] and sex determination (Sox9). In addition, mutational analysis has suggested a role for Sox genes in influencing cell fate decisions during development [Pevny, L. H. and R. Lovell-Badge, Sox genes find their feet. Curr Opin Genet Dev, 1997. 7(3): p. 338-44]. Encoding a 204 amino acid protein, SRY is thought to bind and sharply bend DNA by means of its HMG box to regulate male-specific gene expression [Ferrari, S., V. R. Harley, A. Pontiggia, P. N. Goodfellow, R. Lovell-Badge, and M. E. Bianchi, SRY, like HMG1, recognizes sharp angles in DNA. Embo J, 1992. 11(12): p. 4497-506; Harley, V. R., D. I. Jackson, P. J. Hextall, J. R. Hawkins, G. D. Berkovitz, S. Sockanathan, R. Lovell-Badge, and P. N. Goodfellow, DNA binding activity of recombinant SRY from normal males and XY females. Science, 1992. 255(5043): p. 453-6; King, C. Y. and M. A. Weiss, The SRY high-mobility-group box recognizes DNA by partial intercalation in the minor groove: a topological mechanism of sequence specificity. Proc Natl Acad Sci USA, 1993. 90(24): p. 11990-4; Nasrin, N., C. Buggs, X. F. Kong, J. Camazza, M. Goebl, and M. Alexander-Bridges, DNA-binding properties of the product of the testis-determining gene and a related protein. Nature, 1991. 354(6351): p. 317-20]. The transient expression of Sry during a brief period in the developing genital ridge, between embryonic days E10.5 and E12.5, is what triggers testis development from a bipotential gonad [Koopman, P., A. Munsterberg, B. Capel, N. Vivian, and R. Lovell-Badge, Expression of a candidate sex-determining gene during mouse testis differentiation. Nature, 1990. 348(6300): p. 450-2]. After this strictly regulated window of expression in mouse fetal gonads, Sry is re-expressed in the adult testis. However, while Sry RNA is expressed in the developing genital ridges as a linear transcript of about 5 kb [Hacker, A., B. Capel, P. Goodfellow, and R. Lovell-Badge, Expression of Sry, the mouse sex determining gene. Development, 1995. 121(6): p. 1603-14; Jeske, Y. W., J. Bowles, A. Greenfield, and P. Koopman, Expression of a linear Sry transcript in the mouse genital ridge. Nat Genet, 1995. 10(4): p. 480-2], in adult germ cells Sry RNA is expressed a circular transcript of about 1.3 kb, presumably untranslatable because not associated with ribosomes [Capel, B., A. Swain, S. Nicolis, A. Hacker, M. Walter, P. Koopman, P. Goodfellow, and R. Lovell-Badge, Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell, 1993. 73(5): p. 1019-30].
In addition to the gonads, Sry expression has been discovered in both the adult and embryonic mouse brain. For instance, the hypothalamus and the mesencephalon (midbrain) were both positive for Sry expression in RT-PCR experiments [Lahr, G., S. C. Maxsoni, A. Mayer, W. Just, C. Pilgrim, and I. Reisert, Transcription of the Y chromosomal gene, Sry, in adult mouse brain. Brain Res Mol Brain Res, 1995. 33(1): p. 179-82; Mayer, A., G. Lahr, D. F. Swaab, C. Pilgrim, and I. Reisert, The Y-chromosomal genes SRY and ZFY are transcribed in adult human brain. Neurogenetics, 1998. 1(4): p. 281-8]. Interestingly, the hypothalamus and the mesencephalon are two areas that show functional sex differences [Reisert, I., E. Kuppers, and C. Pilgrim, Sexual differentiation of central catecholamine systems, in Phylogeny and Development of Catecholamine Systems in the CNS of Vertebrates, W. Smeets and A. Reiner, Editors. 1994, Cambridge University Press: Cambridge. p. 453-462; Vadasz, C., G. Kobor, P. Kabai, I. Sziraki, I. Vadasz, and A. Lajtha, Peiinatal anti-androgen treatment and genotype affect the mesotelencephalic dopamine system and behavior in mice. Horm Behav, 1988. 22(4): p. 528-39]. A more comprehensive profile of Sry expression was described using mouse brains as early as embryonic day 11 (E11) through adulthood (postnatal day 90 or P90) [Mayer, A., G. Mosler, W. Just, C. Pilgrim, and I. Reisert, Developmental profile of Sry transcripts in mouse brain. Neurogenetics, 2000. 3(1): p. 25-30]. For all embryonic stages studies, whole brains were obtained for analysis while at postnatal stages, brain regions such as midbrain, diencephalon, and cortex, were isolated. Sry expression was seen at all stages and in all regions so obtained. Particular emphasis was placed on the detection of linear versus circular Sry transcripts in this study [Gonzalez-Hernandez, T. and M. Rodriguez, Compartmental organization and chemical profile of dopaminergic and GABAergic neurons in the substantia nigra of the rat. J Comp Neurol, 2000. 421(1): p. 107-35]. During E11 through E19, Sry transcripts were found to be in the circular untranslatable form. In contrast, postnatal brain Sry transcripts were found to be in the linear (translatable) form suggesting that Sry in the brain is developmentally regulated. This switch in transcript form is directly opposite to the one observed for the gonads. All the expression studies of Sry have been performed by RT-PCR so far, which raises two concerns: (1) since Sry is a single exon gene expressed at low levels, the RT-PCR data are often difficult to interpret, and (2) RT-PCR studies do not provide specific anatomical localization of expression. Further approaches involving in situ hybridization and immunohistochemistry are needed to confirm specific expression of Sry in the adult brain.
The substantia nigra (SN) is a nucleus located in the midbrain that plays a pivotal role in the control of voluntary movement. The SN is cytoarchitecturally divided into three different parts: the SN pars compacta (SNc), the SN pars reticulata, and the SN pars lateralis [Olanow, C. W. and W. G. Tatton, Etiology and pathogenesis of Parkinson's disease. Annu Rev Neurosci, 1999. 22: p. 123-44]. The SNc, a region rich in dopaminergic neurons, has been associated with a prominent human neurological disorder, Parkinson's disease, as dopaminergic neurons of the SNc preferentially degenerate in Parkinson's patients [Castillo, S. O., J. S. Baffi, M. Palkovits, D. S. Goldstein, I. J. Kopin, J. Witta, M. A. Magnuson, and V. M. Nikodem, Dopamine biosynthesis is selectively abolished in substantia nigra/ventral tegmental area but not in hypothalamic neurons in mice with targeted disruption of the Nurr1 gene. Mol Cell Neurosci, 1998. 11(1-2): p. 36-46]. Parkinson's disease is a neurodegenerative disorder caused by SNc dopaminergic cell death and characterized by rigidity, rest tremor, postural instability and bradykinesia. Dopaminergic neurons of the SNc regulate motor function via nigrostriatal projections to the dorsolateral striatum. Transcriptional factors such as β-catenin, Nurr1 and Pitx3 control the differentiation of the DA phenotype [Maxwell, S. L., H. Y. Ho, E. Kuehner, S. Zhao, and M. Li, Pitx3 regulates tyrosine hydroxylase expression in the substantia nigra and identifies a subgroup of mesencephalic dopaminergic progenitor neurons during mouse development. Dev Biol, 2005. 282(2): p. 467-79; Malbon, C. C., Frizzleds: new members of the superfamily of G-protein-coupled receptors. Front Biosci, 2004. 9: p. 1048-58].
Wnts are secreted, lipid anchored proteins [Nusse, R., Wnts and Hedgehogs: lipid-modified proteins and similarities in signaling mechanisms at the cell surface. Development, 2003. 130(22): p. 5297-305] that bind and activate the Frizzled receptor family [Moon, R. T., A. D. Kohn, G. V. De Ferrari, and A. Kaykas, WNT and beta-catenin signalling: diseases and therapies. Nat Rev Genet, 2004. 5(9): p. 691-701]. In the absence of Wnts, β-catenin is phosphorylated by a destruction protein complex including glycogen synthase kinase-GSK3 β, targeting it for ubiquitination and degradation [Zigova, T., A. E. Willing, E. M. Tedesco, C. V. Borlongan, S. Saporta, G. L. Snable, and P. R. Sanberg, Lithium chloride induces the expression of tyrosine hydroxylase in hNT neurons. Exp Neurol, 1999. 157(2): p. 251-8]. Upon Wnt binding to Frizzled receptors, GSK3 β is inhibited, leading to decreased β-catenin phosphorylation (i.e. stabilisation of β-catenin) and accumulation of stabilised β-catenin in the nucleus, where it modulates transcription of TCF/LEF 1 target genes. Lithium chloride (LiCl) treatment of NT2N neurons at therapeutic doses increased the proportion of tyrosine hydroxylase (TH)-positive neurons by six fold after 5 days in culture [Stambolic, V., L. Ruel, and J. R. Woodgett, Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr Biol, 1996. 6(12): p. 1664-8]. LiCl inhibits GSK3 β and thereby stabilises β-catenin [Castelo-Branco, G., N. Rawal, and E. Arenas, GSK-3beta inhibition/beta-catenin stabilization in ventral midbrain precursors increases differentiation into dopamine neurons. J Cell Sci, 2004. 117(Pt 24): p. 5731-7]. In one study, specific inhibitors of GSK3 β increased the size of the DA population ventral mesencephalon neuron cultures by promoting conversion of Nurr1-expressing precursor neurons in TH-positive DA neurons [Becker, J. B., Gender differences in dopaminergic function in striatum and nucleus accumbens. Pharmacol Biochem Behav, 1999. 64(4): p. 803-12]. Therefore mechanisms that lead to stabilisation of β-catenin provide a potential avenue of drug discovery.
Many gender differences in the function of the SNc and its striatal projections have been described and are well summarized by Becker [Saunders-Pullman, R., Estrogens and Parkinson disease: neuroprotective, symptomatic, neither, or both? Endocrine, 2003. 21(1): p. 81-7]. The clinical implications of these differences are apparent in the onset and progression of Parkinson's disease. Males are more susceptible to Parkinson's disease than females, and epidemiologic studies have suggested a role for estrogens in modulating Parkinson's disease (reviewed in [Sherwin, B. B., Estrogen effects on cognition in menopausal women. Neurology, 1997. 48(5 Suppl 7): p. S21−6]). Estrogen administration lowers the severity of Parkinson's disease symptoms in postmenopausal women with early onset of the disease [Fernandez-Ruiz, J. J., M. L. Hernandez, R. de Miguel, and J. A. Ramos, Nigrostriatal and mesolimbic dopaminergic activities were modified throughout the ovarian cycle of female rats. J Neural Transm Gen Sect, 1991. 85(3): p. 223-9]. In animal models, estrogens dramatically alter the function of dopaminergic cells. For example, rat TH and dopamine turnover rate are higher during diestrus (rising estrogen level) than in estrus (low estrogen level) [Ivanova, T. and C. Beyer, Estrogen regulates tyrosine hydroxylase expression in the neonate mouse midbrain. J Neurobiol, 2003. 54(4): p. 638-47]. Estrogen was also shown to regulate TH expression in the mouse midbrain dopaminergic neurons [Dluzen, D., K. Disshon, and J. McDermott, Estrogen as a modulator of striatal dopaminergic neurotoxicity, in Advances in neurodegenerative disorders, Vol. 1, Parkinson's disease, M. J and T. H, Editors. 1998, Prominent: Scottsdale, Ariz.]. Estrogen also has a protective effect against MPTP-induced neurotoxicity in mice [Serova, L. I., S. Mahaijan, A. Huang, D. Sun, G. Kaley, and E. L. Sabban, Response of tyrosine hydroxylase and GTP cyclohydrolase I gene expression to estrogen in brain catecholaminergic regions varies with mode of administration. Brain Res, 2004. 1015(1-2): p. 1-8]. The specific mechanisms by which estrogens act upon dopaminergic neurons are still poorly understood. Short-term injection of estradiol benzoate in rats increased TH mRNA levels in dopaminergic neurons of the SNc, but long-term administration did not [Leranth, C., R. H. Roth, J. D. Elsworth, F. Naftolin, T. L. Horvath, and D. E. Redmond, Jr., Estrogen is essential for maintaining nigrostriatal dopamine neurons in primates: implications for Parkinson's disease and memory. J Neurosci, 2000. 20(23): p. 8604-9]. Consistently, in primates, short-term ovariectomy (10 days) decreased the number of TH-positive cells in the SNc, a reversible effect, whereas long-term ovariectomy (30 days) results in a permanent loss the SNc dopamine cells [Maharjan, S., L. Serova, and E. L. Sabban, Transcriptional regulation of tyrosine hydroxylase by estrogen: opposite effects with estrogen receptors alpha and beta and interactions with cyclic AMP. J Neurochem, 2005. 93(6): p. 1502-14]. The actual transcriptional regulation of TH by estrogen is complex and poorly known, and it seems to depend on the type of estrogen receptors: estrogen increases TH activity with ERalpha but decreases it with ER beta [Castner, S. A., L. Xiao, and J. B. Becker, Sex differences in striatal dopamine: in vivo microdialysis and behavioral studies. Brain Res, 1993. 610(1): p. 127-34]. In addition, there are sex differences in the response of estrogen, as effects seen in females on striatal dopamine release are not seen in males [Dluzen, D. E. and J. L. McDermott, Developmental and genetic influences upon gender differences in methamphetamine-induced nigrostriatal dopaminergic neurotoxicity. Ann N Y Acad Sci, 2004. 1025: p. 205-20], and estrogen functions as a neuroprotectant against metamphetamine in females, but not in males [Canuth, L. L., I. Reisert, and A. P. Arnold, Sex chromosome genes directly affect brain sexual differentiation. Nat Neurosci, 2002. 5(10): p. 9933-4]. More generally, genetically modified mouse models have shown a sex chromosome effect on the number of TH-positive cells cultured from embryonic mesencephalon [Kumer, S. C. and K. E. Vrana, Intricate regulation of tyrosine hydroxylase activity and gene expression. J Neurochem, 1996. 67(2): p. 443-62], suggesting a complex regulation of TH not limited to endocrine influences. In fact, modulation of TH is complex and involves transcriptional control, alternative RNA processing and regulation of RNA stability [Abramham, I. et al., Increased PKA and PKC activities accompany neuronal differentiation of NT2/D1 cells. J. Neurosci. Res., 1991. 28: p. 29 39].
It is proposed that sex differences in the molecular characteristics of brain regions and their associated behaviour are influenced by genetic factors independently of gonadal hormones. The present inventors propose a role for the Y-chromosome encoded testis-determining factor Sry as a transcriptional regulator to increase TH production in DA neurons of the male SN in rodents and humans, and describe a role for Sry in Parkinson's disease.